Geminivirus-based gene expression system

ABSTRACT

A geminivirus based vector system for obtaining controlled expression of a nucleic acid fragment of interest is disclosed. Tissue specific regulatory regions are identified employing cDNA screening and the resulting tissue specific regulatory regions are manipulated for use in geminivirus constructs to provide for transcription and/or expression of nucleic acid sequences nonindigenous to the geminivirus vector for introduction into plant cells. The vector system may be used to provide transformed plants having cells, tissues or parts with a modified phenotypic property.

TECHNICAL FIELD

The present invention relates to the introduction of nucleic acid intoplant cells using a geminivirus-based vector. More particularly, thisinvention relates to use of geminivirus vectors which provide for tissuespecific expression of a transgene in transfected plant cells.

BACKGROUND

For many applications, it is desirable to be able to control geneexpression at a particular stage in the growth of a plant or in aparticular plant cell, tissue or part. For this purpose, methods arerequired which can provide for the desired initiation of transcriptionor expression in the appropriate cell types and/or at the appropriatetime in a plant's development without having serious detrimental effectson plant development and productivity. In general, genetic engineeringtechniques have been directed to modifying the phenotype of individualprokaryotic and eukaryotic cells, especially in culture. Plant cellshave proven more intransigent than other eukaryotic cells, due at leastin part to a lack of suitable vector systems.

The geminiviruses are two-component single-stranded plant DNA viruses.They possess a circular single-stranded (ss) DNA as their genomeencapsidated in twinned "geminate" icosahedral particles. Theencapsidated ss DNAs are replicated through circular double stranded DNAintermediates in the nucleus of the host cell, presumably by a rollingcircle mechanism. Viral DNA replication, which results in thestimulation of both single and double stranded viral DNAs in largeamounts, involves the expression of only a small number of viralproteins that are necessary either for the replication process itself orfacilitates replication or viral transcription. The geminivirusestherefore appear to rely primarily on the machinery of the host to copytheir genomes and express their genes.

Geminiviruses are subdivided on the basis of host range in eithermonocots or dicots and whether the insect vector is a leaf hopper or awhite fly species. The molecular analysis of the genome of an increasingnumber of geminiviruses reinforces this division. All monocot-infectinggeminiviruses are transmitted by leaf hoppers and their genome comprisesa single ss DNA component about 2.7 kb in size; this type of genome, thesmallest known infectious DNA, is typified by wheat dwarf virus which isone of a number from the subgroup that have been cloned and sequenced.By contrast, most members infecting dicot hosts are transmitted by thewhite fly Bemisia tabaci and possess a bipartite genome comprisingsimilarly sized DNAs (usually termed A and B) as illustrated by Africancassava mosaic virus (ACMV), tomato golden mosaic virus (TGMV) andpotato yellow mosaic virus. For successful infection of plants, bothgenomic components are required. Beet curly top virus occupies a uniqueintermediary position between the above two subgroups as it infectsdicots but contains only a single genomic component equivalent to DNA Apossibly as a result of adaption to leaf hopper transmission.

The bipartite subgroup contains only the viruses that infect dicots.Exemplary is the African Cassava Mosaic Virus (ACMV) genome whichcomprises two circular single-stranded DNA molecules each ofapproximately 2.7 kb which contain a homologous region (approximately200 nucleotides) known as the common region. From sequence andmutational analysis, DNA A is known to encode four open reading frames(ORFs). The ORFs are named according to genome component and orientationrelative to the common region, i.e., complementary (c) versus viral (v):AC1, the polymerase gene essential to replication; AC2 is required forvirus spread; AC3, is a regulator of DNA replication; and AV1 is thecoat protein gene. DNA B has two ORFs, BC1 and BV1, both of which arerequired for virus spread. The arrangement of the ORFs shows that theyare expressed in a bidirectional manner. Five major transcripts havebeen identified and these map to the AV1, BV1, BC1, and AC1 ORFs,separately and the AC2/AC3 ORFs together. AC2 has been shown to encode atransacting factor that stimulates production of the coat protein gene,AV1.

Another example from the bipartite group is the tomato golden mosaicvirus (TGMV) which like ACMV is composed of two circular DNA moleculesof the same size, both of which are required for infectivity. Sequenceanalysis of the two genome components reveals six open reading frames(ORFs); four of the ORFs are encoded by DNA A and two by DNA B. On bothcomponents, the ORFs diverge from a conserved 230 nucleotide intergenicregion (common region) and are transcribed bidirectionally from doublestranded replicative form DNA. The ORFs are named according to genomecomponent and orientation relative to the common region (i.e., leftversus right). The AL2 gene product transactivates expression of theTGMV coat protein gene.

There is little sequence analogy between the two DNA components of ACMVand TGMV, except for an almost identical common region of about 200bases, however, the ORFs in the two genomes are analogous and there isthe same requirement for the AL2 gene product for transactivation of thecoat protein gene. Inspection of AL2 sequences from several bipartitegeminiviruses reveal that this protein has the general features expectedof a transacting regulatory protein. It is possible that the requirementfor AL2 function delays coat protein expression for a period of timesufficient to allow dsDNA amplification to occur.

Vectors in which the coat protein ORF has been replaced by aheterologous coding sequence have been developed and the heterologouscoding sequence expressed from the coat protein promoter. However, sinceexpression of the coat protein is dependent upon synthesis of thetransacting regulatory protein, the timing of expression of theheterologous sequence from the coat protein promoter is dependent on thetiming of expression of the transacting regulatory protein. Accordingly,it would be of interest to develop vectors in which the timing of theexpression of the transacting regulatory protein is altered, therebyaltering the timing of expression from the coat protein promoter andthus expression of a heterologous sequence inserted in place of the coatprotein ORF.

The A genome component contains all viral information necessary for thereplication and encapsidation of viral DNA, while the B componentencodes functions required for movement of the virus through theinfected plant. The DNA A component of these viruses is capable ofautonomous replication in plant cells in the absence of DNA B wheninserted as a greater than full length copy into the genome of plantcells, or when a copy is electroporated into plant cells.

Relevant Literature

References relating to geminiviruses include the following: R. H. A.Coutts et al., Aust. J. Plant Physiol. (1990) 17:365-75; Ann Haley etal., Virology (1992) 188:905-909; Garry Sunter et al., The Plant Cell(1992) 4:1321-1331; Clare L. Brough et al., Virology (1992) 187:1-9;Garry Sunter et al., Virology (1991) 180:416-419; and Garry Sunter etal. (1990) Virology (1990) 179:69-77.

Genes which are expressed preferentially in plant seed tissues, such asin embryos or seed coats, have also been reported. See, for example,European Patent Application 87306739.1 (published as 0 255 378 on Feb.3, 1988) and Kridl et al. (Seed Science Research (1991) 1:209-219).

A class of fruit-specific promoters expressed at or during anthesisthrough fruit development, at least until the beginning of ripening, isdiscussed in European Application 88.906296.4, the disclosure of whichis hereby incorporated by reference. cDNA clones that are preferentiallyexpressed in cotton fiber have been isolated. One of the clones isolatedcorresponds to mRNA and protein that are highest during the late primarycell wall and early secondary cell wall synthesis stages. John Crow PNAS(1992) 89:5769-5773. cDNA clones from tomato displaying differentialexpression during fruit development have been isolated and characterized(Mansson et al., Mol. Gen. Genet. (1985) 200:356-361: Slater et al.,Plant Mol. Biol. (1985) 5:137-147).

Mature plastid mRNA for psbA (one of the components of photosystem IIreaches its highest level late in fruit development, whereas after theonset of ripening, plastid mRNAS for other components of photosystem Iand II decline to nondetectable levels in chromoplasts (Piechulla etal., Plant Molec. Biol. (1986) 7:367-376). Recently, cDNA clonesrepresenting genes apparently involved in tomato pollen (McCormick etal., Tomato Biotechnology (1987) Alan R. Liss, Inc., N.Y.) and pistil(Gasser et al., Plant Cell (1989), 1:15-24) interactions have also beenisolated and characterized.

Other studies have focused on genes inducibly regulated, e.g. genesencoding serine proteinase inhibitors, which are expressed in responseto wounding in tomato (Graham et al., J. Biol. Chem. (1985)260:6555-6560: Graham et al., J. Biol. Chem. (1985) 260:6561-6554) andon MRNAS correlated with ethylene synthesis in ripening fruit and leavesafter wounding (Smith et al., Planta (1986) 168: 94-100). Accumulationof a metallocarboxypeptidase inhibitor protein has been reported inleaves of wounded potato plants (Graham et al., Biochem Biophys Res Comm(1981) 101:1164-1170).

Agrobacterium-mediated cotton transformation is described in Urnbeck,U.S. Pat. Nos. 5,004,863 and 5,159,135 and cotton transformation byparticle bombardment is reported in WO 92/15675, published Sep. 17,1992. Transformation of Brassica has been described by Radke et al.(Theor. Appl. Genet. (1988) 75;685-694; Plant Cell Reports (1992)11:499-505.

Transformation of cultivated tomato is described by McCormick et al.,Plant Cell Reports (1986) 5:81-89 and Fillatti et al., Bio/Technology(1987) 5:726-730.

SUMMARY OF THE INVENTION

Novel recombinant geminivirus constructs including expression cassettesand transfer vectors are provided which allow for controlledtranscription and/or expression in a transfected plant cell of proteinsnonindigenous to the geminivirus. Also provided are methods of makingthe expression cassettes and methods of using them to producetransfected plant cells having an altered genotype and/or phenotype. Theexpression cassettes include a transactivatable expression cassette anda transacting expression cassette which may be combined in a binaryplasmid. The transactivatable expression cassette has as operably linkedcomponents a transcription initiation unit obtainable from a geminiviruscoat protein gene, a nucleic acid fragment other than a full lengthcoding sequence of the geminivirus coat protein gene, and atranscription termination region. The transacting expression cassettehas as operably linked components a transcription initiation unitobtainable from a 5' non-coding region of a gene which is expressedother than constitutively in one or more plant cell types, tissues, orparts, a DNA fragment comprising a coding sequence from a geminivirusgene encoding a transacting regulatory protein and a transcriptiontermination region. A transfected plant cell may be produced bycontacting a plant cell with a recombinant geminivirus transfer vector,where the transfer vector may comprise a combination of the transactingand the transactivatable expression cassettes. Alternatively, acombination of transfer vectors may be used, for example, a geminivirushaving a genome in which the equivalent of the ACMV AC2 gene of DNA Ahas been inactivated and at least a portion of the coding sequence ofthe mat protein gene in the genome has been replaced with a nucleic acidfragment nonindigenous to the geminivirus and the 5' non-coding regionof the equivalent of the ACMV AC2 gene of DNA B has been replaced with anucleic acid fragment which in combination with the coat protein 5'non-coding region comprises a transcription unit; and a transactingexpression cassette.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows construction of a geminivirus based binary plasmid.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In accordance with the subject invention, novel recombinant geminivirusconstructs including transfer vectors and methods for making them andusing them are described. When used to transfer a plant cell thetransfer vectors provide transgenic plant cell capable of controlledtranscription or expression of nucleic acid fragment in one or moreplant cell types. Depending upon the nature of the regulatory sequencesused in the vector, transcription or expression of the nucleic acidfragment can occur in one or more particular cell types, such as seed orembryo cells or such as pollen grains or cells derived from plantovaries, such as cotton fibers, as compared with other plant cells; inresponse to a particular stimulus, such as wounding of the plant, forexample by insect attack, or an environmental stress such as heat orhigh salinity. Plants in which particular tissues and/or plant partshave an altered phenotype may be produced by the subject method.

The constructs include several forms, depending upon the intended use ofthe construct. Thus, the constructs include vectors, trancriptionalcassettes, expression cassettes and binary plasmids. Two basicconstructs are required which then may be combined in a variety of waysfor transfecting a plant cell and obtaining a transgenic plant cellcapable of controlled transcription or expression of a nucleic acidfragment. These two constructs include a transactivatable expressioncassette and a transacting expression cassette.

The transactivatable expression cassette has as operably linkedcomponents a transcription initiation unit obtainable from a geminiviruscoat protein gene, a nucleic acid fragment other than a full lengthcoding sequence of the geminivirus coat protein gene, and atranscription termination region. The expression cassette can beprepared by replacing the native coding region of the coat protein genewith a nucleic acid fragment which is other than a full length codingsequence of the geminivirus coat protein gene, particularly a codingsequence nonindigenous to the geminivirus which is the source of thecoat protein gene.

The "transcription initiation unit" comprises a geminivirus genomefragment obtainable from the 5' non-coding region of a coat protein genewherein the fragment is of a size and nucleic acid sequence sufficientto provide for transcription of the operably linked nucleic acidfragment following activation by a geminivirus transactivatingregulatory protein. By "obtainable" is intended a transcriptioninitiation unit having a DNA sequence sufficiently similar to that of anative geminivirus coat protein transcription initiation unit to providefor transcription of the operably linked nucleic acid fragment followingactivation by a geminivirus transactivating regulatory protein whichactivates transcription from the native sequence. Obtainable includesboth natural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. The term "nonindigenouscoding sequence" means a coding sequence which does not occur in theunmodified genome of the specific geminivirus which is used forpreparation of compositions of the claimed invention. A modifiedgeminivirus coding sequence, whether modified by mutation, truncation,joining to other sequences, and the like, including a sequence otherthan a full length coding sequence constitutes a nonindigenous codingsequence.

The transacting expression cassette has as operably linked components atranscription initiation unit obtainable from a 5' non-coding region ofa gene which is expressed other than constitutively in one or more plantcell types, plant tissues, or plant parts; a DNA fragment comprising acoding sequence from a geminivirus gene encoding a transactingregulatory protein or a congener thereof having the same or similartransactivating activity; and a transcription termination region.

For transfection, the-transactivatable expression cassette and thetransacting cassette may be combined to form a binary plasmid or the twoexpression cassettes may be introduced into a cell on separate plasmids.By "a gene which is expressed other than constitutively" is intended agene transcription of which is controlled, either positively ornegatively, as to time, for example relative to a stage of tissuedevelopment such as preanthesis to fruit ripening in an ovary cell,and/or tissue of transcription, for example preferentially in fruit ascompared to leaves.

The transfer vector is prepared by making a further modification to theviral genome. The coding sequence of the coat protein gene is replacedwith a restriction site into which a nucleic acid fragment fortranscription or expression may be cloned. This transfer vector can becombined with the transacting cassette on a binary vector or beintroduced into cells on separate plasmids.

A further autonomously replicating transfer vector can be constructedfrom a modified geminivirus in which the coding region for the coatprotein is replaced by a restriction site into which a nucleic acidfragment for transcription or expression may be cloned, and in which a5'-noncoding region from a gene which is expressed other thanconstitutively is inserted preferably 3' to the AC1 coding region and 5'to the AC2 coding region to allow for controlled expression of the AC2gene. For expression of an active AC2, resconstruction of the part ofthe AC2 coding region encompassed in the AC1 coding region may berequired. The autonomously replicating vector may be transferred intoplants alone or as part of a binary vector.

As a source for a modified geminivirus genome for preparation of anautonomously replicating geminivirus transfer vector or for geminivirusDNA fragments comprising a transacting regulatory protein codingsequence or a transcriptional initiation unit from a coat protein gene,any geminivirus genome may be used. The geminivirus genome fragmentsused for preparation of the transacting and trans-activatable expressioncassettes may be from the same virus or different viruses so long as thetransactivating regulatory protein and the coat protein transcriptionunit are capable of interacting to provide for transcription of thenucleic acid fragment operatively linked to the coat proteintranscription unit. Examples of suitable geminiviruses included ACMV,TGMV, potato yellow mosaic virus, BGMV, beet curly top and squash leafcurl. Harrison (1985) Ann. Rev. Phytopath. 23:55-82.

The transactivating expression cassette provides for controlledexpression of the transacting regulatory protein by use of a sufficientportion of a 5' non-coding region obtainable from a gene which isexpressed other than constitutively in a plant cell to provide forexpression of the coding sequence for the transacting regulatory proteinto which it is operably linked. The transcriptional and translationalinitiation region (also sometimes referred to as a "promoter,")preferably comprises a transcriptional initiation regulatory region anda translational initiation regulatory region of untranslated 5'sequences, "ribosome binding sites," responsible for binding mRNA toribosomes and translational initiation. It is preferred that all of thetranscriptional and translational functional elements of the initiationcontrol region are derived from or obtainable from the same gene. Insome embodiments, the promoter will be modified by the addition ofsequences, such as enhancers, or deletions of nonessential and/orundesired sequences. By "obtainable" is intended a promoter having a DNAsequence sufficiently similar to that of a native promoter to providefor the desired specificity of transcription of a DNA sequence ofinterest. It includes natural and synthetic sequences as well assequences which may be a combination of synthetic and natural sequences.

The regulatory regions are capable of directing controlled expression ofa geminivirus transacting regulatory protein. By "controlled expression"is intended expression that occurs in one or a few types of plant cells,and not at all or at low levels in other cells. The expression may be asa result of a developmental change or an external stimulus or otherchange which results in the turning on or off of expression ofparticular genes in a limited number of cell types. A promoter whichdirects expression in a cell such as an ovary cell from anthesis throughflowering but directs little or no expression after the initial changeswhich occur at the time surrounding pollination and/or fertilization orin other plant tissues is an example of a regulatory region capable ofdirecting controlled expression. Other examples include a promoter whichdirects expression in leaf cells following damage to the leaf, forexample from chewing insects but directs little or no expression inother tissues, transcriptional regulatory regions from patatin as anexample for modification of expression tubers, and promoters that directincreased expression in response to environmental stimuli such asincreased salinity of the agrisphere, and the like. In some embodiments,it will be desired to selectively regulate expression in a particulartissue or tissues. For example, selective regulation of expression inseed tissue, including embryo and seed coat tissue is desired formodification of seed products, including seed oils, starch, and storageproteins. For seed oil modification, a variety of phenotype alterationsare of interest. These include modifying the fatty acid composition ofseed oil, such as changing the ratio and/or amounts of the various fattyacids, as to chain length, degree of saturation, and the like. Theseresults can be achieved by providing for reduction of expression of oneor more endogenous products, particularly enzymes or cofactors, byproducing a transcription product which is complementary to thetranscription product of a native gene, so as to inhibit the expressionof the gene product, or providing for expression of a gene, eitherendogenous or exogenous, associated with fatty acid synthesis. Ofparticular interest are transcriptional initiation regions associatedwith storage proteins, such as napin, cruciferin, β-conglycinin,phaseolin, zein or oil bodies, such as oleosin, or genes involved infatty acid biosynthesis, including acyl carrier protein (ACP),stearoyl-ACP desaturase, and fatty acid synthases, and other genesexpressed during embryo development, such as Bce4. For example, the ACPpromoter provides an appropriate timing pattern for fatty acidbiosynthesis modification, and the methods described herein may be usedto increase the level of transcription or expression of a desired geneproduct over that particular period of ACP expression.

For example, when used in conjunction with a 5' untranslated sequencecapable of initiating translation, expression in defined ovary tissue,including ovary integuments (also known as "ovule epidermal cells"),core or pericarp tissue, and the like, the transcriptional initiationregion can direct a desired message encoded by a DNA sequence ofinterest in a particular tissue to more efficiently effect a desiredphenotypic modification. For example, expression in ovary pericarptissue, also known as the ovary wall and/or ovary core tissue, couldresult in useful modifications to the edible portions of many fruits,including true berries such as tomato, grape, blueberry, cranberry,currant, and eggplant; stone fruits (drupes), such as cherry, plum,apricot, peach, nectarine and avocado; and compound fruits (druplets),such as raspberry and blackberry. In hesperidium (oranges, citrus), suchexpression cassettes are expected to be expressed in the "juicy" portionof the fruit. In pepos, (such as watermelon, cantaloupe, honeydew,cucumber, and squash) the equivalent tissue is most likely the inneredible portions. In other fruits, such as legumes, the equivalent tissueis the seed pod.

The modification of analogous structures of non-edible fruit may also beof interest. Thus, of special interest are transcription initiationregions expressible in at least ovary outer pericarp tissue. Forexample, in cotton the analogous ovary structure is the burr of thecotton boll, in rapeseed it is the seed pod. In a like manner,regulating expression in ovary integuments and/or core tissue may resultin useful modifications to the analogous fruit and related structuresevolving therefrom, for example seed coat hairs, such as cotton fibers.Cotton fiber is a differentiated single epidermal cell of the outerintegument of the ovule. It has four distinct growth phases; initiation,elongation (primary cell wall synthesis), secondary cell wall synthesis,and maturation. Initiation of fiber development appears to be triggeredby hormones. The primary cell wall is laid down during the elongationphase, lasting up to 25 days postanthesis (DPA). Synthesis of thesecondary wall commences prior to the cessation of the elongation phaseand continues to approximately 40 DPA, forming a wall of almost purecellulose. In addition to ovary tissue promoters, transcriptionalinitiation regions from genes expressed preferentially in seed tissues,and in particular seed coat tissues, are also of interest forapplications where modification of cotton fiber cells is considered.

An example of a gene which is expressed at high levels in Brassica seedcoat cells is the EA9 gene described in EPA 0 255 378. The nucleic acidsequence of a portion of the EA9 cDNA is provided therein, and can beused to obtain corresponding sequences, including the promoter region.An additional seed gene which is expressed in seed embryo and seed coatcells is the Bce4 Brassica gene. The promoter region from this gene alsofinds use in the subject invention; this gene and the correspondingpromoter region are described in WO 91/13980, which was published Sep.19, 1991. Fiber specific proteins are developmentally regulated. Thus,transcriptional initiation regions from proteins expressed in fibercells are also of interest. An example of a developmentally regulatedfiber cell protein, is E6 (John and Crow Proc. Nat. Acad. Sci.(USA)(1992) 89:5769-5773). The E6 gene is most active in fiber, althoughlow levels of transcripts are found in leaf, ovule and flower.

To obtain a specifically derived transcriptional initiation region, thefollowing steps may be employed. Messenger RNA (mRNA) is isolated fromtissue of the desired developmental stage. This mRNA is then used toconstruct cDNA clones which correspond to the mRNA population both interms of primary DNA sequence of the clones and in terms of abundance ofdifferent clones in the population. mRNA is also isolated from tissue ofa different developmental stage in which the target gene should not beexpressed (alternate tissue). Radioactive cDNA from the desired tissueand from the alternate tissue is used to screen duplicate copies of thecDNA clones. The preliminary screen allows for classification of thecDNA clones as those which correspond to mRNAs which are abundant inboth tissues; those which correspond to mRNAs which are not abundant ineither tissue; those which correspond to mRNAs which are abundant in onetissue and relatively non-abundant in the other. Clones are thenselected which correspond to mRNAs that are abundant only in the desiredtissue and then these selected clones are further characterized.

Since the hybridization probe for the preliminary screen outlined aboveis total cDNA from a particular tissue, it hybridizes primarily to themost abundant sequences. In order to determine the actual level ofexpression, particularly in tissue where the mRNA is not as abundant,the cloned sequence is used as a hybridization probe to the total mRNApopulation(s) of the desired tissue(s) and various undesired tissue(s).This is most commonly done as a Northern blot which gives informationabout both the relative abundance of the mRNA in particular tissues andthe size of the mRNA transcript.

It is important to know whether the abundance of the mRNA is due totranscription from a single gene or whether it is the product oftranscription from a family of genes. This can be determined by probinga genomic Southern blot with the cDNA clone. Total genomic DNA isdigested with a variety of restriction enzymes and hybridized with theradioactive cDNA clone. From the pattern and intensity of thehybridization, one can distinguish between the possibilities that themRNA is encoded either by one or two genes or by a large family ofrelated genes. It can be difficult to determine which of severalcross-hybridizing genes encodes the abundantly expressed mRNA found inthe desired tissue. In this case it is important to use a probe which iscapable of distinguishing a particular gene from the remainder of thefamily members.

The cDNA obtained as described can be sequenced to determine the openreading frame (probable protein-coding region) and the direction oftranscription so that a desired target DNA sequence later can beinserted at the correct site and in the correct orientation into atranscription cassette. Sequence information for the cDNA clone alsofacilitates characterization of corresponding genomic clones includingmapping and subcloning as described below. At the same time, a genomiclibrary can be screened for clones containing the complete gene sequenceincluding the control region flanking the transcribed sequences. Genomicclones generally contain large segments of DNA (approximately 10-20 kb)and can be mapped using restriction enzymes, then subcloned andpartially sequenced to determine which segments contain thedevelopmentally regulated gene.

Using the method described above, several transcriptional regulatoryregions have been identified. One example is the tomato-derivedtranscriptional initiation region which regulates expression of thesequence corresponding to the pZ130 cDNA clone. Sequences hybridizableto the pZ130 clone, for example, probe pZ7, show abundant mRNA,especially at the early stages of anthesis. The message is expressed inovary integument and ovary outer pericarp tissue and is not expressed,or at least is not readily detectable, in other tissues or at any otherstage of fruit development. Thus, the pZ130 transcriptional initiationregion is considered ovary-specific for purposes of this invention. ThepZ130 transcriptional initiation region is described in U.S. Pat. No.5,175,095, which disclosure is incorporated herein by reference.

A promoter from a tomato gene, referred to as 2A11, was isolated usingthe methods described above. The 2A11 promoter provides for an abundantmessenger, being activated at or shortly after anthesis and remainingactive until the red fruit stage. Expression of the 2A11 gene under the2A11 promoter occurs only in fruit; generally no detectable expressionis obtained in root, leaves or stems. The gene encodes a sulfur-richamino acid sequence similar to plant storage proteins in sulfur contentand size. The 2A11 transcriptional initiation region is described inU.S. Pat. No. 4,943,674 issued Jul. 24, 1990 and in PCT applicationWO90/04063 which disclosures are hereby incorporated by reference.

Tissue-specific transcription suggests that gene regulatory proteins maybe bound to enhancer sequences and other upstream promoter elements inspecific cells. By enhancer element ("enhancer") is intended aregulatory DNA sequence that is capable of activating transcription froma promoter linked to it with synthesis beginning at the normal RNA startsite; which is capable of operating in both orientations (normal orflipped); and which is capable of functioning even when moved eitherupstream or downstream from the promoter. Both enhancers and otherupstream promoter elements bind sequence specific DNA-binding proteinsthat mediate their effects.

As an example, to identify the exact nucleotide sequences important forthe function of the enhancer(s), and other upstream elements, fragmentsof the 2A11 5'-region are screened for their capacity to bind nuclearproteins and for their ability to function in a heterologous promoter.Binding experiments with nuclear proteins from fruit-tissue and othertissue such as leaf can be used to determine the presence of enhancerand silencer sequences; the protein binding studies can be used topinpoint specific nucleotide sequences that bind to a correspondingseries of gene regulatory proteins.

The activity of each enhancer and other upstream promoter elementsgenerally is present on a segment of DNA which may contain binding sitesfor multiple proteins. The binding sites can generally be dissected bypreparing smaller mutated versions of the enhancer sequence joined to areporter gene whose product is easily measured, such as the Gus gene.The effect of each mutation on transcription can then be tested.Alternatively, fragments of this region can be prepared. Each of themutated versions of the enhancer sequence or the fragments can beintroduced into an appropriate host cell and the efficiency ofexpression of the reporter gene measured. Those nucleotides required forenhancer function in this test are then identified as binding sites forspecific proteins by means of gel mobility shift and DNA footprintingstudies. An alternate means of examining the capability of the isolatedfragments to enhance expression of the reporter gene is to look forsub-domains of the upstream region that are able to enhance expressionlevels from a promoter which comprises the TATA CAAT box but showslittle or no detectable activity. An example of such a promoter is thetruncated 35S promoter (see for example Poulsen and Chua, Mol. Gen.Genet. (1988) 214:16-23 and Benfey, et al., EMBO J. (1990) 9:1677-1684and 1685-1696 and Gilmartin, Plant Cell (1990) 2:369-378). A fragment ofthe 5'-region is inserted in front of the truncated promoter in anexpression cassette, and the effect on expression of the reporter geneevaluated. PCT application WO90/04063 which disclosure is herebyincorporated by reference discloses how to make and how to use upstreamregulatory sequences as exemplified using the 2A11 gene.

Other promoter regions of interest include those which regulateexpression of the enzyme polygalacturonase, an enzyme which plays animportant role in fruit ripening. The polygalacturonase promoter isactive in at least the breaker through red fruit stage. In determiningoptimum amounts of other 5' regions, such as that from the PG gene,which are required to give expression of a DNA sequence of interestpreferentially in fruit, screening can be carried out as described abovefor the 2A11 5' region, using a reporter gene such as Gus. Thepolygalacturonase gene is described in U.S. Pat. No. 4,535,060 issuedAug. 13, 1985, U.S. Pat. No. 4,769,061 issued Sep. 6, 1988, U.S. Pat.No. 4,801,590 issued Jan. 31, 1989 and U.S. Pat. No. 5,107,065 issuedApr. 21, 1992, which disclosures are incorporated herein by reference.

Also of interest are 5' non-coding regions from the following genes:elongation factor EF-1, which is active in meristematic tissue, and isdisclosed in U.S. Pat. No. 5,177,010 issued Jan. 5, 1993; MAC, which isdisclosed in U.S. Pat. No. 5,106,739 which issued Apr. 21, 1992; heatshock, disclosed in U.S. Pat. No. 5,187,267 issued Feb. 16, 1993; andACC/napin which is active in seed and is disclosed in EP 255 378. Thecited patents and applications are incorporated herein by reference.

A transcriptional transactivatable cassette will include as operablylinked components in the direction of transcription, geminivirus coatprotein transcriptional initiation region and optionally a translationalinitiation region, a nucleic acid sequence of interest, and atranscriptional and optionally a translational termination regionfunctional in a plant cell. When the cassette provides for thetranscription and translation of a nucleic acid sequence of interest itis considered an expression cassette. One or more introns may be also bepresent in the cassette. Other sequences may also be present in thetransactivatable cassette, including those encoding transit peptides andsecretory leader sequences as desired. How to obtain and use thesesequences are well known to those skilled in the art.

Downstream from, and under the regulatory control of, the coat proteintranscriptional/translational initiation control region is a nucleotidesequence of interest. The nucleotide sequence may be any open readingframe encoding a polypeptide of interest, for example, an enzyme, or asequence complementary to a genomic sequence, where the genomic sequencemay be an open reading frame, an intron, a noncoding leader sequence, orany other sequence where the complementary sequence inhibitstranscription, messenger RNA processing, for example, splicing, ortranslation. The nucleotide sequences of this invention may besynthetic, naturally derived, or combinations thereof. Depending uponthe nature of the nucleotide sequence of interest, it may be desirableto synthesize the sequence with plant preferred codons. The plantpreferred codons may be determined from the codons of highest frequencyin the proteins expressed in the largest amount in the particular plantspecies of interest.

Phenotypic modification can be achieved by modulating production eitherof an endogenous transcription or translation product, for example as tothe amount, relative distribution, or the like, or an exogenoustranscription or translation product, for example to provide for a novelfunction or products in a transgenic host cell or tissue. Of particularinterest are DNA sequences encoding expression products associated withthe development of plant fruit, including genes involved in metabolismof cytokinins, auxins, ethylene, abscissic acid, and the like. Methodsand compositions for modulating cytokinin expression are described inU.S. Pat. No. 5,177,307, which disclosure is hereby incorporated byreference. Alternatively, various genes, from sources including othereukaryotic or prokaryotic cells, including bacteria, such as those fromAgrobacterium tumefaciens T-DNA auxin and cytokinin biosynthetic geneproducts, for example, and mammals, for example interferons, may beused. Other genes of interest include fatty acid biosynthesis genesincluding medium and long-chain thioesterases (WO 91/16421 publishedOct. 31, 1991), desaturase (WO 91/13972 published Sep. 19, 1991),synthases (WO 92/03564, published Mar. 5, 1992); flavor genes such asSPS (WO 91/19808, published Dec. 26, 1991); and ripening genes, such asanti sense polygalacturonase (U.S. Pat. No. 4,801,540), anti-senseethylene genes such as ACCD, ACC synthase, ethylene forming enzyme (EFE)(WO 91/02958). Other phenotypic modifications include modification ofthe color of plant parts developing from ovary integuments and/or coretissue, for example seed coat hairs, such as cotton fibers. Of interestare genes involved in production of melanin and genes involved in theproduction of indigo. Melanins are dark brown pigments found in animals,plants and microorganisms, any of which may serve as a source forsequences for insertion into the constructs of the present invention.Color in cotton and color and fruit quality may be modified by the useof carotenoid pathway genes (EP 0 393 690 and WO 91/13078).

Transactivatable transcriptional cassettes may be used when thetranscription of an anti-sense sequence is desired. When the expressionof a polypeptide is desired, transactivatable expression cassettesproviding for transcription and translation of the nucleotide sequenceof interest will be used. Various changes are of interest; these changesmay include modulation (increase or decrease) of formation of particularsaccharides, hormones, enzymes, or other biological parameters. Thesealso include modifying the composition of the final fruit or fiber, forexample changing the ratio and/or amounts of water, solids, fiber orsugars. Desirable seed modifications includ oil content, fatty acidcomposition, and storage protein content. Other phenotypic properties ofinterest for modification include response to stress, organisms,herbicides, brushing, growth regulators, and the like. These results canbe achieved by providing for reduction of expression of one or moreendogenous products, particularly an enzyme or cofactor, either byproducing a transcription product which is complementary (anti-sense) tothe transcription product of a native gene, so as to inhibit thematuration and/or expression of the transcription product, or byproviding for expression of a gene, either endogenous or exogenous, tobe associated with the development of a particular tissue or plant part.

The termination region which is employed in the transactivatable andtransacting expression cassettes will be primarily those which are ofconvenience, since the termination regions appear to be relativelyinterchangeable. The termination region which is used may be native withthe transcriptional initiation region, may be native with the DNAsequence of interest, may be derived from another source. Thetermination region may be naturally occurring, or wholly or partiallysynthetic. Convenient termination regions are available from theTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase termination regions. In some embodiments, it may be desired touse the 3' termination region native to the transcription initiationregion used in a particular construct.

The various constructs normally will be joined to a marker for selectionin plant cells. Conveniently, the marker may be resistance to a biocide,particularly an antibiotic, such as kanamycin, G418, bleomycin,hygromycin, chloramphenicol, or the like. The particular marker employedwill be one which will allow for selection of transformed cells ascompared to cells lacking the DNA which has been introduced. Componentsof DNA constructs including transcription cassettes of this inventionmay be prepared from sequences which are native (endogenous) or foreign(exogenous) to the host. By foreign is intended that the sequence is notfound in the wild-type host into which the construct is introduced.Heterologous constructs will contain at least one region which is notnative to the gene from which the transcription initiation region isderived.

In preparing the constructs, the various DNA fragments may bemanipulated, so as to provide for DNA sequences in the properorientation and, as appropriate, in proper reading frame for expression;adapters or linkers may be employed for joining the DNA fragments orother manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. In vitro mutagenesis, primer repair, restriction,annealing, resection, ligation, or the like may be employed, whereinsertions, deletions or substitutions, e.g. transitions andtransversions, may be involved. Conveniently, a vector or cassette mayinclude a multiple cloning site downstream from the ovary-relatedtranscription initiation region, so that the construct may be employedfor a variety of sequences in an efficient manner.

In carrying out the various steps, cloning is employed, so as to amplifythe amount of DNA and to allow for analyzing the DNA to ensure that theoperations have occurred in proper manner. By appropriate manipulations,such as restriction, chewing back or filling in overhangs to provideblunt ends, ligation of linkers, or the like, complementary ends of thefragments can be provided for joining and ligation. A wide variety ofcloning vectors are available, where the cloning vector includes areplication system functional in E. coli and a marker which allows forselection of the transformed cell. Illustrative vectors include pBR332,pUG series, M13mp series, pACYC184, etc. Thus, the sequence may beinserted into the vector at an appropriate restriction site(s), theresulting plasmid used to transform the E. coli host, the E. coli grownin an appropriate nutrient medium and the cells harvested and lysed andthe plasmid recovered. Analysis may involve sequence analysis,restriction analysis, electrophoresis, or the like. After eachmanipulation the DNA sequence to be used in the final construct may berestricted and joined to the next sequence. Each of the partialconstructs may be cloned in the same or different plasmids.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a plant cell host. Thesetechniques include transfection with DNA employing A. tumefaciens or A.rhizogenes as the transfecting agent, protoplast fusion, injection,electroporation, particle acceleration, etc. For transformation withAgrobacterium, plasmids can be prepared in E. coli which contain DNAhomologous with the Ti-plasmid, particularly T-DNA. The plasmid may ormay not be capable of replication in Agrobacterium, that is, it may ormay not have a broad spectrum prokaryotic replication system such asdoes, for example, pRK290, depending in part upon whether thetranscription cassette is to be integrated into the Ti-plasmid or to beretained on an independent plasmid. The Agrobacterium host will containa plasmid having the vir genes necessary for transfer of the T-DNA tothe plant cell and may or may not have the complete T-DNA. At least theright border and frequently both the right and left borders of the T-DNAof the Ti- or Ri-plasmids will be joined as flanking regions to thetranscription construct. The use of T-DNA for transformation of plantcells has received extensive study and is amply described in EPA Ser.No. 120,516, Hoekema, In: The Binary Plant Vector SystemOffset-drukkerij Kanters B. V., Alblasserdam, 1985, Chapter V, Knauf, etal., Genetic Analysis of Host Range Expression by Agrobacterium, In:Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. ed.,Springer-Verlag, N.Y., 1983, p. 245, and An, et al., EMBO J. (1985)4:277-284.

For infection, particle acceleration and electroporation, a disarmedTi-plasmid lacking particularly the tumor genes found in the T-DNAregion) may be introduced into the plant cell. By means of a helperplasmid, the construct may be transferred to the A. tumefaciens and theresulting transfected organism used for transfecting a plant cell;explants may be cultivated with transformed A. tumefaciens or A.rhizogenes to allow for transfer of the transcription cassette to theplant cells. Alternatively, to enhance integration into the plantgenome, terminal repeats of transposons may be used as borders inconjunction with a transposase. In this situation, expression of thetransposase should be inducible, so that once the transcriptionconstruct is integrated into the genome, it should be relatively stablyintegrated. Transgenic plant cells are then placed in an appropriateselective medium for selection of transgenic cells which are then grownto callus, shoots grown and plantlets generated from the shoot bygrowing in rooting medium.

To confirm the presence of the transgenes in transgenic cells andplants, a Southern blot analysis can be performed using methods known tothose skilled in the art. Expression products of the transgenes can bedetected in any of a variety of ways, depending upon the nature of theproduct, and include immune assay, enzyme assay or visual inspection,for example to detect pigment formation in the appropriate plant part orcells. Once transgenic plants have been obtained, they may be grown toproduce plant tissues or parts having the desired phenotype. The planttissue or plant parts, may be harvested, and/or the seed collected. Theseed may serve as a source for growing additional plants with tissues orparts having the desired characteristics. The terms transgenic plantsand transgenic cells include plants and cells derived from eithertransgenic plants or transgenic cells.

The various sequences provided herein may be used as molecular probesfor the isolation of other sequences which may be useful in the presentinvention, for example, to obtain related transcriptional initiationregions from the same or different plant sources. Related 5'non-codingregions obtainable from the sequences provided in this invention willshow at least about 60% homology, and more preferred regions willdemonstrate an even greater percentage of homology with the probes. Ofparticular importance is the ability to obtain related transcriptioninitiation control regions having the timing and tissue parametersdescribed herein. For example, using the probe pZ130, at least 7additional clones, have been identified, but not further characterized.Thus, by employing the techniques described in this application, andother techniques known in the art (such as Maniatis, et al., MolecularCloning; A Laboratory Manual (Cold Spring Harbor, N.Y.) 1982), othertranscription initiation regions capable of controlled direction oftranscription and/or expression as described in this invention may bedetermined. The constructs can also be used in conjunction with plantregeneration systems to obtain plant cells and plants; the constructsmay also be used to modify the phenotype of plant tissues and plantparts produced thereby.

The invention finds particular use in controlling transcription orexpression of a nucleic acid fragment so as to provide for preferentialor at least substantially specific expression in a particular tissue,for example in fruit or seeds as compared to other tissues such as leaf,stems or roots. By "at least substantially" is intended that expressionof a nucleic acid fragment of interest in the particular tissue ortissues or parts is greater by about 100 fold the expression of thenucleic acid fragment in other tissues or parts of the plant. By "fruit"is intended the ripened ovary wall of a flower and any other closelyassociated parts. (See Weirer, T. E. et al., ed., Botany: AnIntroduction to Plant Biology (6th ed.) (John Wiley & Sons, 1982);Tootill & Backmore, eds., The Facts on a File Dictionary of Botany(Market Home Boob Ltd., 1984). The following examples are offered by wayof illustration and not by limitation.

EXAMPLES EXAMPLE 1 Preparation of ACMV Expression Cassettes

The complete nucleotide sequences of ACMV (formerly called CLV) DNA 1and DNA 2 are published (Stanley and Gay, 1983, Nature 301,260-262). Toconstruct a binary vector for plant transformation containing the viraltransacting factor AC2 under the control of the ACP regulatory elements,a C12-specific thioesterase from bay laurel under the control of thevital coat protein regulatory elements and a kanamycin resistance geneunder the control of the CaMV 35s promoter, the following constructs aremade.

The coat protein (AV1) regulatory sequences (labeled CP5' and CP3') wereobtained by PCR using the following oligonucleotides and pETA092 as atemplate. pETA092 (obtained from J. Stanley, John Innes Inst., Norwich,UK), is a complete clone of ACMV DNA 1 in the Mlu1 site of the cloningvector pIB120 (International Biotechnologies, Inc., New Haven, Conn.).The oligonucleotides for the coat protein 5' were #3558,5'-CTGGAGCTCATGTFGACCAAGTCAATFGG-3'(SEQ ID NO. 1) and #3560,5'-GCTACTAGTGGATCCCACATTGCGC-3'(SEQ ID NO. 2) and were designed toamplify the ACMV DNA 1 sequences between nucleotides 2752 and 299 whichincludes the common region and the coat protein transcription startsite. Oligonucleotide #3558 incorporates a Sac1 restriction site at the5'-end of the PCR fragment and oligo #3560 incorporates a Spe1 site atthe 3'-end of the fragment for subcloning. The 3'oligonucleotides were#3564, 5'CCACTGCAGCGACGTTGAAAATACG-3'(SEQ ID NO. 3) and 3559,5'-CACACTAGTCAATGTAATTAGAGCTGC-3'(SEQ ID NO. 4). They were designed toamplify ACMV DNA1 from nucleotides 1175 to 1315 with a Pst1 and Spe1site on the 5' and 3'-ends of the fragment respectively. This fragmentcontains the potential polyadenylation signal for the coat protein gene.The PCR conditions were: 94 degrees Centigrade, 10 min, 72 degreesCentigrade, 7 min, for addition of the Taq polymerase, then thirtycycles of 94 degrees Centigrade for 15 sec, 50 degrees Centigrade for 30sec, and 72 degrees Centigrade for 30 sec.

The coat protein 3' sequences were subcloned from the PCR reaction(described above) to create pCGN3289 by cutting the PCR DNA with Pst 1and Spe 1 and ligation to a modified chloramphenicol resistant pCGN565(pCGN3288) also cut with Pst1 and Spe1. pCGN3288 was constructed bydigestion of pCGN565 (described in other patent applications) withHindIII and Pst1 and ligation of a synthetic linker containing HindIII,Spe1 and Pst1 sites. The linker was made by annealing the syntheticoligonucleotides 5'-AGCTTCCACTAGTGGCTGCA-3'(SEQ ID No. 5) and5'-GCCACTAGTCCA-3'(SEQ ID NO. 6). Bay laurel thioesterase codingsequence was isolated from pCGN3826 (described in CGN82-4WO patentapplication) by a BamH1 complete and Pst 1 partial digestion. Theisolated 1.27 kb thioesterase sequence was then cloned upstream of thecoat protein 3 by ligation of the isolated thioesterase fragment withpCGN3289 digested with BamH1 and Pst1. The plasmid containing thethioesterase sequences and the CP3' is named pCGN3291.

The coat protein 5'-sequence was subcloned from the PCR DNA by digestionwith SacI and Spe1 and ligation to a modified Bluescript II KS(-) vector(Stratagene, La Jolla, Calif.) named pCGN3290 cut with Sac1 and Spe1.pCGN 3290 was made by digestion of pBluescript II KS(-) with BAMH1 andSpe1 and ligation with pCGN3291 cut with BamH1 and Spe1. The resultingplasmid contains an expression cassette consisting of the CP5', thethioesterase gene, and the CP 3' in an ampicillin resistant backbone.

The AC2 open reading frame was obtained by PCR using primers with thefollowing sequences: 5'-TGCTGAATTCAGAATGCAATCTFCATCACCC-3'(SEQ ID NO. 7)and 5'-TGCTCTGCAGCTAAAGACCCTTAAGAAAAGACC-3'(SEQ ID NO. 8) correspondingto nucleotides 1774 to 1754 surrounding the ATG start codon of the AC2open reading frame and nucleotides 1386 to 1364 surrounding the stopcodon. The primers have EcoR1 and Pst1 sites respectively on the endsfor further cloning steps. The template for the PCR was 30 cycles, then10 min at 72 degrees Centigrade. The resulting PCR fragment (410 bp) wassubcloned into pBluescript II SK(-) by digestion with EcoR1 and Pst1.The AC2 open reading frame is inserted in the ACP expression cassette,pCGN1977 (Soberer et al., 1992, Plant Molecular Biology 18, 591-594), bydigestion with EcoR1 and Pst1 and ligation to pCGN1977 cut with EcoR1and Pst1.

The CP 5'/TE/CP 3' cassette is combined with the ACP5'/AC2/ACP3'cassette by respective digestion with Sac1 and Spe1 andAsp718 and Sac1 and ligation of the plasmids with an Asp718 and Xbal cutbinary vector such as pC2GN1557 (McBride and Summerfelt, 1990, PlantMolecular Biology 14, 269-276). The resulting binary vector contains:the left border--35s/NPT11/tm1-ACP5'/AC2/ACP3 -CP 5'/TE/CP3' rightborder expression cassette in a gentamicin resistant background. Thebinary vector can be transformed into Agrobacterium tumefaciens and usedto produce Brassica plants.

The above invention relates to the use of geminivirus vectors to providefor controlled expression of a nucleic acid fragment by using the5'coding region from genes which provide for developmental regulation inplant cells in place of the native promoter of the geminivirus geneencoding a transacting regulatory protein. In this way, thetranscription cassettes and expression cassettes can be produced whichallow for differentiated cell production of the desired product from thecoat protein promoter of the geminivirus. Thus, the phenotype of aparticular plant part may be modified, without requiring that theregulated product be produced in all tissues, which may result invarious adverse effects on the growth, health, and productioncapabilities of the plant. Particularly, tissue specific transcriptioninitiation capability is provided for modifying the phenotypicproperties of a variety of tissues.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic oligonucleotide                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      CTGGAGCTCATGTTGACCAAGTCAATTGG29                                               (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic oligonucleotide                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      GCTACTAGTGGATCCCACATTGCGC25                                                   (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic oligonucleotide                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CCACTGCAGCGACGTTGAAAATACG25                                                   (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic oligonucleotide                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      CACACTAGTCAATGTAATTAGAGCTGC27                                                 (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic oligonucleotide                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      AGCTTCCACTAGTGGCTGCA20                                                        (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic oligonucleotide                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      GCCACTAGTCCA12                                                                (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic oligonucleotide                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      TGCTGAATTCAGAATGCAATCTTCATCACCC31                                             (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: synthetic oligonucleotide                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      TGCTCTGCAGCTAAAGACCCTTAAGAAAAGACC33                                           __________________________________________________________________________

What is claimed is:
 1. A geminivirus transfer vector comprising a geminivirus genome wherein the coding sequence for the coat protein gene in said genome is deleted and replaced with a first multiple cloning site and wherein the transcriptional initiation region of the geminivirus transacting factor gene in said genome is replaced with a second multiple cloning site.
 2. The vector of claim 1, wherein a DNA sequence encoding a transcriptional initiation region of a gene which is expressed other than constitutively in one or more plant cells is inserted into said second multiple cloning site.
 3. The vector of claim 1, wherein a DNA sequence of interest is inserted into said first multiple cloning site.
 4. The vector of claim 1, wherein said geminivirus genome is repeated at least 1.2 times.
 5. The vector of claim 1, wherein said geminivirus is an African cassava mosaic virus.
 6. A plant cell comprising a geminivirus transfer vector of claim
 1. 7. The vector of claim 3, wherein expression of said DNA sequence of interest modifies a plant phenotype.
 8. The vector of claim 3, wherein a transcription product of said DNA sequence of interest modifies a plant phenotype.
 9. The vector of claim 4, further comprising a right T-DNA border.
 10. A transacting expression cassette comprising as operatively linked components in the 5' to 3' direction of transcription:a transcriptional initiation region obtained from a 5' non-coding region of a plant gene which is expressed other than constitutively in one or more plant cells;a DNA fragment encoding a geminivirus coat protein transacting factor; and a transcriptional termination region.
 11. The transacting expression cassette of claim 10, wherein said plant gene which is expressed other than constitutively is a gene coding for a polypeptide selected from the group consisting of:ACP, Bce4, napin, polygalacturonase, 2A11, pZ7, hsp80, EF-1, ssu, and EA9.
 12. The transacting expression cassette of claim 10, wherein said geminivirus is an African cassava mosaic virus.
 13. A plant cell comprising a transacting expression cassette of claim
 10. 14. A binary plasmid comprising:(1) A transactivatable cassette comprising a transcription initiation region obtained from a geminivirus coat protein gene; a DNA sequence of said coat protein gene, the expression of which modifies a plant phenotype; and a transcription termination region; and (2) a transacting expressing cassette comprising a transcriptional initiation region obtained from a 5' non-coding region of a plant gene which is expressed other than constitutively in one of more plant cells; a DNA fragment encoding a coat protein transacting factor; and a transcriptional termination region.
 15. The binary plasmid of claim 14, further comprising a right T-DNA border.
 16. The binary plasmid of claim 14, further comprising a plant selectable marker.
 17. A plant cell comprising a binary plasmid of claim
 14. 18. A binary vector comprising:(1) a geminivirus genome wherein a coding sequence in a coat protein gene in said genome is deleted and replaced with a multiple cloning site and a DNA sequence encoding a geminivirus transacting factor is modified to prevent said geminivirus from producing a functional transacting factor protein; (2) a transacting expression cassette comprising a transcriptional initiation region obtained from a 5' non-coding region of a plant gene which is expressed other than constitutively in one or more plant cells, a DNA fragment encoding a geminivirus coat protein transacting factor and a transcriptional termination region; and (3) a right T-DNA border region.
 19. A method of producing a geminivirus transfer vector, said method comprising:in a geminivirus genome, replacing a coding sequence of a coat protein gene with a DNA sequence of interest, the expression of which, modifies a plant phenotype and replacing the transcriptional initiation region of the geminivirus transacting factor gene in said genome with a DNA sequence encoding a transcriptional initiation region of a gene which is expressed other than constitutively in one or more plant cells. 